The Effect Of Chitosan Derivatives On The Growth Of Corneal Cells

Corneal transplantation is an effective way for treating corneal blindness, however, there are many problems, such as a serious shortage of donor cornea, immune rejection and other postoperative problems. With the development of tissue engineering technology, tissue engineering cornea opens up new prospects for corneal defect treatment. The core is to establish the composition of three-dimensional complex with cells and biological materials and the key is the choice of scaffold material and cells cultured in vitro. Up to now, materials used for corneal tissue engineering scaffold are collagen, amniotic membrane, acellular corneal stroma, fibrin and chitosan. But there are short comings, limiting the development and application with those materials.Chitosan is a natural polycationic bio-polysaccharide, biodegradable and non-toxic. It has been greatly used in research and application of corneal tissue engineering. However, the biocompatibility of chitosan can not satisfide the needs of eye implantation, limiting its development. The modified chitosan can be much better than chitosan in the aspect of biocompatibility as corneal tissue engineering scaffold materials. Scaffold materials implanted in the body are degraded into low molecular chitosan derivatives. The security and functionality of these derivatives on corneal cells are also important researches.In the topic, chitosan is modified by chemical method and enzymatically degraded into a variety of derivatives. It is carried out to determinate the physical and chemical properties, toxicity, materials compatibility on corneal cells in turn. The results provide theoretical basis for corneal biological safety of scaffold material.The experimental results and progress are as followed:1. Chitosan is modified by inserting functional groups into chitosan molecules according to the chemical preparation methods in the laboratory, obtained four kinds of derivatives (Carboxymethyl chitosan, Carboxymethyl chitin, Hydroxyethyl chitosan and Hydroxypropyl chitosan).Then they are degraded by enzyme into small molecular derivatives (Carboxymethyl chitin oligosaccharide, Carboxymethyl chitosan oligosaccharide and Chitooligosaccharides) and monosaccharide (N-acetyl-glucosamine and Glucosamine Hydrochloride). The physical and chemical properties of the materials are determined. In order to evaluate cell toxicity of materials, the combination of microscopic observation and MTT assay is used. The results are that there is no cell toxicity of all these materials. Carboxymethyl chitin, Hydroxyethyl chitosan, Chitooligosaccharides, and Carboxymethyl chitin oligosaccharide can promote cell growth and proliferation of L929 cells. It provides a theoretical support for following experiments。2. The corneal epithelial cells are cultured by conventional tissue-adherent method. A large number of cells with high activity are prepared for the evaluation of materials. The effect of materials on corneal epithelial cells is evaluated by microscopic observation and MTT assay. The results show that there is no toxicity of the four kinds of polysaccharides (Hydroxyethyl chitosan, Hydroxypropyl chitosan, Carboxymethyl chitosan and Carboxymethyl chitin). Hydroxyethyl chitosan and Carboxymethyl chitin have a good role in promoting the growth of the epithelial cells. It suggests that these four kinds of water-soluble chitosan derivatives (Hydroxyethyl chitosan, Hydroxypropyl chitosan, Carboxymethyl chitosan and Carboxymethyl chitin) can be safely used as scaffolds for corneal epithelium. The degraded materials with low molecular are non-toxic on the corneal epithelial cells. Chitooligosaccharides and Carboxymethyl chitin oligosaccharide can significantly promote the proliferation of epithelial cells, suggesting that both of them can be used as medium additives for corneal epithelial cells.3. Corneal stromal cells with higher activity are cultured in vitro for evaluating materials.The results show that nine kinds of chitosan derivatives are not toxic on corneal stromal cells, suggesting that materials have good biocompatibility. Low molecular saccharides, such as N-acetyl-glucosamine, Chitooligosaccharides, Carboxymethyl chitin oligosaccharide and polysaccharides, such as Chondroitin Sulfate, Carboxymethyl chitin, can promote the growth and proliferation of stromal cells. It suggests that N-acetyl-glucosamine, Chitooligosaccharides, Carboxymethyl chitin oligosaccharide can be used in the culture of corneal stromal cells. As Chondroitin Sulfate and Carboxymethyl chitin promote cell growth and proliferation, so they are conducive to the corneal defect and reconstruction. Chitosan derivatives prepared in the experiment can be used safely for tissue engineering cornea. It provides the necessary theoretical asis for the following study of corneal tissue engineering.